Snf1 protein kinase of Saccharomyces cerevisiae is a member of the highly conserved Snf1/AMP-activated protein kinase (AMPK) family, which plays a central role in responses to metabolic stress. In humans, AMPK regulates glucose and lipid metabolism to maintain cellular energy balance and has been implicated in the pathogenesis of human disease, including diabetes, obesity, cancer, and hereditary heart disease. In yeast, the Snfl pathway has a primary role in the response to nutrient stress. We propose to continue our functional analysis of the Snfl/AMPK pathway in the yeast system, which offers the advantages of powerful genetics and has informed our understanding of the human pathway. We propose genetic and biochemical studies to address the roles of the Snf1-activating kinases (Pak1, Tos3, and Elm1) and the Reg1-Glc7 protein phosphatase 1 in regulating Snft catalytic activity. The proposed work on the noncatalytic subunits of Snf1 protein kinase, Snf4 and Gal83, will elucidate their functions in regulating the activity and localization of the protein kinase. Snf4 comprises domains that are involved in AMP binding in AMPK. The Gal83 beta subunit controls the glucose-regulated nuclear localization of the kinase. Studies of the interconnections between the Snf1 pathway and the highly conserved PKA, Yak1, and CK2 pathways will provide insight into the role of the Snf1/AMPK pathway in cellular regulation. Finally, we propose to take advantage of the yeast system for genetic analysis of mammalian LKB1, which is a major activating kinase for AMPK and AMPK-related kinases and has important roles in suppressing tumorigenesis. Mutations in LKB1 in humans cause hereditary Peutz-Jeghers cancer syndrome. LKB1 alone, or in the LKB1-STRAD- MO25 complex, functions as a Snf1-activating kinase in yeast, thereby allowing selection of mutations that improve its activating function. The proposed studies will elucidate regulatory mechanisms that control the AMPK pathway, which is important in type 2 diabetes, obesity, cancer, and hereditary heart disease. These studies will provide insight into human disease and lead to development of drugs to activate AMPK for treatment. The proposed work on mammalian LKB1, a tumor suppressor that activates AMPK, will provide clues for the design of activating drugs.